DESCRIPTION (Verbatim from Applicant's abstract): For two decades, it has been known that smooth muscle has the unique capacity to regulate both cross bridge activation, and the rate that cross-bridges cycle and hydrolyze ATP. As a result, during sustained contractions, smooth muscles utilize very little chemical energy. The molecular mechanism for regulation of this unique "latch-state" remains unknown in spite of the central role of vascular smooth muscle in hypertension and other cardiovascular diseases. The proposed studies will challenge the current paradigm, that activation of smooth muscle actomyosin requires the covalent phosphorylation of the myosin regulatory light chain, by testing a model for thin-filament linked regulation, which may significantly advance our understanding of how the latch-state is regulated. These studies will specifically investigate the role of caldesmon, an actin and tropomyosin binding protein, as a modulator of thin filament-linked regulation. These studies will test the hypothesis that caldesmon modulates the transition of regulated thin filaments from a turned-off state to a turned-on state that cooperatively activates cross-bridge cycling. This controversial hypothesis is the direct outcome of the development and application by the applicant's laboratory of a modified in vitro motility assay that allows the applicant to measure changes in the force exerted by myosin on individual regulated thin filaments that have been reconstituted from purified and expressed smooth muscle proteins. Using this assay, in conjunction with protocols developed during the current funding period to fully turn-on smooth muscle thin filaments, the first specific aim of this proposal will demonstrate that caldesmon specifically blocks the activation of both unphosphorylated and phosphorylated smooth muscle myosin by turned-on thin filaments. The second specific aim will test the hypothesis that phosphorylation of caldesmon by MAP kinase reverses the inhibitory effects of caldesmon on thin filament regulation of myosin, and will identify the active sites of phosphorylation by using reconstituted thin filaments containing either caldesmon that has been phosphorylated by MAP kinase, or expressed caldesmon fragments in which specific phosphorylation sites have been mutated to aspartic acid to mimic phosphorylation. Overall, these studies should significantly advance the understanding of the regulation of smooth muscle contraction, and will shed new light on the regulatory role of caldesmon, as well as the molecular processes that regulate the "latch-state" in smooth muscle.